E4:

4.1 Techniques in modern Biotechnology

4.2 Applications in Biotechnology

4.3 Chapter 1: Technique in Modern Biotechnology

Outline -Recombinant DNA technology steps -Advantages of recombinant DNA -Genetically modified organisms -Plant steps - Animal Cloning -Advantages & disadvantages of cloning -Polymerase chain reaction

● Recombinant DNA technology steps: 1. Obtain DNA fragments containing of interest using restriction enzymes

2. Obtain vectors (e.g. : small rings of extrachromosomal DNA)

3. Cut DNA fragments and plasmids using same restriction enzymes(restriction) ->restriction enzyme recognizes the specific base sequence and cuts the double-stranded DNA to produce sticky ends, which are paired together once again by complementary base pairing 4. Join DNA fragments and plasmids together using DNA ligase (ligation) ->catalyse the joining of DNA fragments and plasmids by covalent bonds

5. Transfer the DNA into a host , those possessing the selective gene marker can be identified and separated ● Advantages of recombinant DNA: 1. Synthetic hormones not rejected by immune system 2. Products are pure 3. Product yield is higher (growth rate of bacteria is high, can continuously produce products) 4. Extraction cost is low

●Genetically modified organisms (GMOs): using recombinant DNA technology to infect host cells so the organism can develop specific traits

Benefits of GMOs: 1. Large amount of useful products produced in a short time and at a low cost 2. Increase food supplies 3. Higher nutritional value in GM food 4. Reduce pollution

Hazards of GMOs: 1. Long term effects on human health is unknown 2. Transfer altered to wild types () 3. Out compete wild types, reduce biodiversity and upset ecological balance ● Plant cloning steps: 1. Plant tissues sterilized and put into sterile culture medium containing all nutrients necessary for growth 2. A mass of undifferentiated cells called the is formed from mitotic 3. Transfer the callus to a culture medium with hormones to promote shoot and root growth 4. Plantlets are formed and transferred to soil for further growth

Applications and benefits of plant cloning: 1. Produce large number of plants in a short time 2. Produce disease-free plants 3. Produce genetically identical plants 4. Maintain special breed of plants 5. Produce plants that are endangered or hard to grow

● Animal cloning ○ splitting: embryo at 6-cell stage split to individual cells and develop into genetically identical individuals by separating them using a sterilized knife

○ Nuclear transfer/ cell nuclear transfer (SCNT): 1. Mammary gland cell obtained 2. Ovum collected from another individual but with nucleus removed by 3. Mammary gland cell fused with ovum with no nucleus and develop into an embryo by development 4. Embryo developed from the cell is stimulated by electric shock was implanted into the uterus of a surrogate mother 5. An animal is produced

Applications of animal cloning: 1. Propagate farm animals or endangered animals 2. Produce genetically identical animals 3. Obtain stem cells 4. Mass produce GM animals

Limitations of animal cloning: 1. Success rate is low 2. Clones get old sooner and have shorter life span ● Advantages of cloning: preserve observable traits and overcome reproductive difficulties

● Disadvantages of cloning: Lack genetic variations and reduce adaptability to changes in the environment

● Polymerase chain reaction(PCR): Uses: rapid gene amplification without living cells, all reagents placed in the thermal cycler

1. DNA denaturation (95°C): DNA is denatured and the double helix of the DNA sample separates into 2 single DNA strands

2. Annealing of primers (50-65°C): primers (primers are short synthetic single stranded DNA to act as starting points for DNA synthesis) anneal to single stranded DNA templates by complementary base pairing by ‘bracketing’ the DNA segment to be amplified

3. DNA synthesis/Extension stage (70°C): Heat stable DNA polymerase catalyse the addition of free nucleotides to the new DNA strand

Copies of DNA=2*n where n=number of cycles

Applications of PCR: 1. Diagnosis of genetic diseases 2. DNA fingerprinting 3. Identify remains of historical figures or extinct species 4. Diagnose infectious diseases Chapter 2: Applications in Biotechnology

Outline -Vaccine - -Bacterial vectors. -Applications of stem cells -Bacteria as host cell. -Limitation of stem cells -

● Human insulin/human growth insulin/vaccines/ monoclonal antibodies

● Bacterial vector as : Gene 1. Pick up foreign DNA easily 2. Picked up by host cells due to their small size 3. Usually carries marker genes (antibiotic i resistance gene) to allow selection of transformed cells Plasmid ● bacteria as host cell: 1. Transform easily Plasmids 2. Can grow in inexpensive culture media 3. Reproduce quickly 4. Easily extracted and purified FEE. Bacterial DNA ● Gene therapy gene therapy (not-inheritable): supplement a defective gene with a normal one

Ex vivo (outside): 1. Cells removed from body (isolate defect cells and isolate normal genes then amplify with PCR) 2. Vectors with normal genes transfer the genes into cells 3. Cells return to body

In vivo (inside): vectors with normal genes are directly involved into the body Ex vivo Vs. In vivo

Injection Transgenic QQ.io:*. → packaging → into virus Therapeutic To p gene Patients

Stem cells removed from patients O Viral translation in the lab

→ i ← pop . *÷smo.** Patients Therapeutic gene IRetreatment of transduced cells

Benefits of gene therapy: Hazards of gene therapy 1. Treat genetic diseases 1. Cause severe immune responses 2. Treat and 2. Viral vector may regain ability to infectious diseases cause disease 3. Insertion of genes affect expression of existing genes ● Stem cell therapy: Undifferentiated cells Embryonic Adult that can carry out 00Stem Cell Stem Cell unlimited mitotic cell division 8 8 1. : Pluripotent come from Stem Cell Multipotent and can develop into 00Stem Cell almost any cell types

2. Adult stem cells: come 8 8 from marrow, skeletal muscles and Any Cell Limited peripheral blood and can 00Type develop into limited range of cells

● Applications of stem cells:

1. Cell research (understand how cells divide and differentiate to develop treatments) 2. Drug toxicity tests 3. Diseases treatments ->Somatic cells: shorter effects, somatic cells constantly replaced by new cells ->Stem cells: longer effects, stem cells carrying normal gene divide to form more stem cells and differentiate into new cells ● Limitations of stem cells: 1. Adult stem cells can’t be identified 2. Conditions for culturing stem cells to differentiate into different tissues are unknown 3. Isolation of embryonic stem cells destroys 4. Stem cells may keep on dividing to cause cancer Chapter 3: Bioethics

Outline -GM food -Stem cell therapy -Cloning - project - Gene Therapy

●GM food

Purpose: -improve yield: resist plant viruses, insects and herbicides -enhancing nutritional contents .

Detrimental effects: 1. Long term effects on humans are unknown ⇒⇐⇒ 2. Cause allergic reactions f¥¥EBg*¥* 3. Lead to ‘superbugs’ 4. Out-compete wild types and reduce GM food biodiversity 5. Genetic pollution 6. Toxins accumulate along food chain

● Cloning

1. Low success rate 2. Expensive ⑥ aao t t 3. Decrease genetic •• variations among ① population

4. Destruction of embryo ÷ when obtaining nucleus t :÷÷÷÷÷i¥ t

, onto Cloning

● Gene therapy 1. Hard to decide when to use 2. Widening social class gap 3. Large sum of money required

→ Therapeutic Therapeutic

Transgene • u / • Target cell • ● Stem cell therapy 1. Destruction of human lives 2. The right to decide the future of embryos

1,7¥. ÷ & 7¥ #8: -

Stem cell therapy

● 1. Termination of foetus with defective gene 2. Owner of genetic information is unknown 3. Possible discrimination for people’s genes 4. Affect public perception of an individual